北京地区2000—2004年分离的呼吸道合胞病毒B亚型株G蛋白基因的序列分析

Sequence analysis of G glycoprotein of human respiratory syncytial virus subtype B strains isolated from children with acute respiratory infections in Beijing, China in years 2000-2004

目的 了解近年来北京地区流行的B亚型呼吸道合胞病毒（BSV）G蛋白的基因特征。方法 2000年冬季至2004年冬季自首都儿科研究所附属儿童医院收集的急性呼吸道感染患儿呼吸道标本，从中分离到B亚型RSV毒株，每年随机选取1至2株毒株，用RT-PCR扩增其G蛋白全基因后克隆至pBS-T载体中，筛选出阳性克隆后测序，并与B亚型标准株（CH18537）和文献报道的毒株序列进行比较分析。结果 所测毒株G蛋白全基因核苷酸长度分别为915、921和981bp，推导的氨基酸长度分别为292、293、312和315从。分离株与B亚型标准株CH18537间存在着明显的差异，CH18537株同分离株间的核苷酸的同源性为91．9％～93．7％，氨基酸的同源性只有85．0％～89．0％。分离株间核苷酸的同源性为93．4％～98．8％，氨基酸的同源性为88．2％～98．6％。氨基酸的变异主要集中在胞外区一个高度保守区的两端，而胞内区和跨膜区相对保守。此外，在进行G蛋白基因分析的8株B亚型分离毒株中，我们发现有3株BSVG蛋白在490～495位核苷酸缺失，3株RSVG蛋白在c末端791位后出现了60个核苷酸的插入，导致C末端259位后出现20个氨基酸的插入。这60个核苷酸与相邻的前60个核苷酸高度重复，只出现3至4个核苷酸的差异。该3株分离株与文献报道的有60个核苷酸插入的两株（S00-4和BA4128／99B）核苷酸和氨基酸同源性分别为97．5％～98．6％和95．5～98．1％，在插入的20个氨基酸中，有高达50％（9～10个氨基酸）左右的丝氨酸和苏氨酸氧连接的糖基化位点。结论 RSVB亚型G蛋白基因变异存在着多样性，分离株既有核苷酸替代，又有基因缺失和插入，还有糖基化位点的改变和氨基酸长度的改变。这种变异使G蛋白高度糖基化，提示最终可能导致病毒抗原性的改变。北京分离的有60个核苷酸插入的变异株与日本及西班牙报道的变异株有非常近的亲缘关系，提示这种G蛋白基因中有60个核苷酸插入的B亚型变异株已经在子代病毒中形成稳定遗传并在世界范围内传播。

Objective To characterize the G glycoprotein gene of human respiratory syncytial virus （RSV） subtype B isolated from children in Beijing in recent years. Methods Eight RSV subtype B strains used for analyzing were isolated from clinical samples collected from infants and children with acute respiratory infections and visited the Children＇s Hospital affiliated to Capital Institute of Pediatrics, Beijing during the period of winter of 2000 to winter of 2004. One to two strains for each year were picked up randomly. The full length of G protein encoding genes from those strains was amplified by RT-PCR from the virus isolates and then cloned into pBS-T vector. The clones with target G protein encoding genes were identified by PCR and the inserts were then sequenced and compared with those subtype B strains published and the prototype strain CH18537. Results The G genes from those isolates were 915, 921 and 981 bp in length with predicted amino acid of 292,293, 312 and 315 AA. The identities of nucleotides and amino acids among these RSV B subtype isolates in Beijing from 2000 to 2004 and prototype strains CH18537 were 91.2%-93.4% and 85%-89%, respectively. The identifies of nucleotides and amino acids among these RSV B subtype isolates in Beijing were 93.1%-98.9% and 88.2%-98.6%, respectively. The divergence of amino acids occurred mainly in the proposed extracellular domain with a highly conserved region in between. The intracellular domain and cross membrane domain are conserved. There were nucleotide deletions at 490-495 in 3 out of those 8 strains. In 3 out of those 8 strains, there were 60 nucleofides insertion after nt 791 at the C terminal of the genes, which leads to 20 AA insertions after AA 259 at the C terminal of the proteins. These insertions were highly repeated with the 60 nucleotides in front border upon, with only 3 to 4 nucleotides differences. The identifies of nucleotides and amino acids of the G protein genes of these 3 strains to those G protein genes with 60 nucleotides insertion（strain S（D-4 and BA4128/99B）published previously were 97.5%-98.6% and 95.5- 98.1%, respectively. Among those 20 inserted amino acids, there were as high as 50% （9 to 10 amino acids） serine and threonine linked glycosylated sites. Conclusion Sequence analysis revealed that the G protein encoding genes of subtype B RSV strains isolated in Beijing in recent years were highly diverse, with nucleotide deletion, insertion, substitution, the change of the glycosylate sites as well as the variation of the length of the amino acids. These variations make the G proteins from those strains highly glycocylated, suggest that the antigenieity of these proteins may be changed. The G protein genes with 60 nucleotide insertions from those isolates in Beijing were close to those isolates from Japan and Spain, suggest that those strains with the 60 nucleotide insertions have circulated world wide.